Use of osteoprotegerin (OPG) to increase human pancreatic beta cell survival and proliferation

ABSTRACT

It is disclosed herein that osteoprotegerin increases human beta cell proliferation and survival. Methods are provided for increasing beta cell proliferation, but contacting a beta cell with an effective amount of osteoprotegerin, a functional fragment, variant or fusion protein thereof. Methods are also provided for treating a human subject with diabetes, comprising administering to the subject a therapeutically effective amount of osteoprotegerin, functional variant or fusion protein thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.13/802,159, filed Mar. 13, 2013, which claims the benefit of U.S.Application No. 61/613,356, filed Mar. 20, 2012. The prior applicationsare incorporated by reference herein in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. DK072264awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

This relates to the field of diabetes, specifically to methods forincreasing human beta cell survival and proliferation.

BACKGROUND

A mammalian pancreas is composed of two subclasses of tissue: theexocrine cells of the acinar tissue and the endocrine cells of theislets of Langerhans. The exocrine cells produce digestive enzymes thatare secreted through the pancreatic duct to the intestine. The isletcells produce polypeptide hormones that are involved in carbohydratemetabolism. The islands of endocrine tissue that exist within the adultmammalian pancreas are termed the islets of Langerhans. Adult mammalianislets are composed of five major cell types, the α, β, δ, PP, and εcells. These cells are distinguished by their production of glucagon,insulin, somatostatin, pancreatic polypeptide, and ghrelin,respectively.

Diabetes mellitus results from the failure of cells to transportendogenous glucose across their membranes either because of anendogenous deficiency of insulin or an insulin receptor defect. Diabetestype 1 is caused by the destruction of β cells, which results ininsufficient levels of endogenous insulin. Diabetes type 2, may initiateas a defect in either the insulin receptor itself or in the number ofinsulin receptors present or in the balance between insulin and glucagonsignals, although it is ultimately caused due to a loss of functional βcells. Current treatment of individuals with clinical manifestation ofdiabetes attempts to emulate the role of the pancreatic β cells in anon-diabetic individual. Individuals with normal β cell function exhibitprecise regulation of insulin secretion in response to serum glucoselevels. This regulation is due to a feedback mechanism that resides inthe β cells that ordinarily prevents surges of blood sugar outside ofthe normal limits. Unless blood sugar is controlled properly, dangerousor even fatal levels can result. Hence, treatment of a diabeticindividual involves monitoring of blood glucose levels and the use ofinjected bovine, porcine, or cloned human insulin as required. Despitesuch intervention, there is often a gradual decline in the health ofdiabetics. Diabetes afflicts millions of people in the United Statesalone, and there is a clear need to provide additional treatments forthis disease.

SUMMARY

It is disclosed herein that osteoprotegerin (OPG) increases beta cellproliferation and survival, specifically human beta cell proliferationand survival. Methods are provided for increasing beta cellproliferation, by contacting a beta cell with an effective amount ofOPG, a functional fragment or variant thereof, or a fusion proteinthereof, such as an Fc fusion protein.

In some embodiments, methods are provided for treating a subject withdiabetes or pre-diabetes, comprising administering to the subject atherapeutically effective amount of OPG, or a functional fragment or avariant thereof, or an Fc Fusion protein thereof. In other embodiments,methods are provided for treating a subject with diabetes orpre-diabetes, comprising administering to the subject a therapeuticallyeffective amount of a nucleic acid encoding OPG, or a functionalfragment thereof, a variant thereof, or an Fc Fusion protein thereof. Insome embodiments, the subject is human. In yet other embodiments, thesubject has type I diabetes.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of OPG. (adapted from Holen I, Shipman CM. Role of osteoprotegerin (OPG) in cancer. Clin Sci (Lond).110:279-291, 2006).

FIGS. 2A-2E shows lactogens regulate OPG. Ratio of OPG/actin (internalcontrol) mRNA in islets from normal (NL) and RIP-mPL1 transgenic (TG)mice cultured in serum-free medium at 5.5 mM glucose for 24 hours (h)(n=4-5 each) by (FIG. 2A) apoptosis PCR array from SABiosciences, and(FIG. 2B) real-time PCR. (FIG. 2C) Prl (200 ng/ml) treatment for 24 hincreases OPG/actin mRNA ratio compared to vehicle (veh) treated INS-1cells by real-time PCR (n=6). (FIG. 2D, FIG. 2E) Preliminary westernblot analysis of OPG and tubulin expression in NL and RIP-mPL1 TG mouseislets (FIG. 2D), or INS-1 cells treated ±Prl for 24 h (FIG. 2E) (n=2).

FIGS. 3A-3C are a schematic diagram and a set of bar graphs. FIG. 3Ashows the molecular partners of OPG. These graphs show expression of themRNA encoding receptors RANK and Death Receptor (DR) in mouse islets andINS-1 cells with and without lactogens by real time PCR.

FIGS. 4A-4C show bromodeoxyuridine (BrdU)-positive β-cells in the threegroups of mice. FIG. 4A is a digital image of an immunohistochemicalstain showing BrdU (red) and Insulin (green). The 50 ng dose of OPGresulted in a significant increase in β-cell proliferation (n=3 each) inmice. FIG. 4B shows the effect of dose on proliferation in a 7-daystudy. FIG. 4C shows the effect of dose on β cell proliferation in a 30day study. Veh=vehicle control.

FIGS. 5A-5C shows that OPG enhances human β cell proliferation. FIG. 5Ashows co-staining of human islet cell cultures (BrdU (red), insulin(green) and DAPI (blue)). FIG. 5B is a bar graph showing the effect ofdifferent doses of human osteoprotegerin (hOPG) or vehicle (veh) onβ-cell replication in human islet cell cultures treated for 24 h.Quantification of the percentage of BrdU-positive β-cells in thedifferent conditions shows that the 50, 100 and 200 ng/ml doses of hOPGsignificantly increased β-cell proliferation versus veh-treated cells(n=4-8 preps each in duplicate/triplicate; * p<0.05). FIG. 5C is a bargraph showing that OPG at 100 ng increased β cell proliferation whenevaluated using Ki67.

FIGS. 6A-6C (FIG. 6A) Human β-cell death measured by insulin (red),TUNEL (green), and DAPI (blue) co-staining under GLT conditions for 36 hin vehicle (veh) and hOPG (200 ng/ml) treated human islet cell cultures.(FIG. 6B) Quantification of TUNEL-positive β-cells in control (Ctrl) andGLT conditions, relative to Veh-Ctrl β-cell death as 100. GLT caused amarked increase in β-cell death in veh-treated cells which wassignificantly reduced with OPG treatment (n=4 preps, in duplicate).(FIG. 6C) Quantification of TUNEL-positive β-cells in control (Ctrl) andcytokine-treated cells, depicted as fold-over Veh-Ctrl. Cytokines(interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and interferon(IFN)-γ) caused a marked increase in β-cell death in veh-treated cellswhich was significantly reduced with different doses of hOPG treatment(n=5; 3 human islet preps, in singlicate/duplicate) * p<0.05 vsveh-ctrl; # p<0.05 vs veh-cytokines.

FIG. 7 is a schematic diagram of a treatment protocol.

FIGS. 8A-8B Four treatment groups for the mice (FIG. 8A); experimentaldesign (FIG. 8B), depicting time of various treatments. BrdU is injected6 h before sacrifice at which time pancreas and blood is collected.

FIG. 9 Percent diabetic NOD/Ltj female mice injected daily with saline,starting at 10-weeks of age with treatment continuing up to 21-weeks ofage. Blood glucose was measured weekly, and mice with glucose values>250mg/dl were considered diabetic (n=8).

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file[8123-88743-03_Sequence_Listing.txt, Feb. 26, 2016, 5.67 KB], which isincorporated by reference herein.

The nucleic and amino acid sequences are shown using standard letterabbreviations for nucleotide bases, and three letter code for aminoacids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

SEQ ID NO: 1 is the amino acid sequence of human osteoprotegerin.

SEQ ID NO: 2 is an exemplary nucleic acid sequence encoding humanosteoprotegerin.

DETAILED DESCRIPTION

Diabetes results from a reduction in the endogenous functional β-cellmass. It is now known that despite this loss, residual β-cells arepreserved in the pancreas of patients with diabetes, including peoplewith long-term diabetes. Therefore, an approach to the treatment andcure of both Type 1 and Type 2 diabetes has been to find ways to expandthe remnant β-cell mass, such as by increasing β cell proliferation.Recent studies in rodents have shown irrefutably that adult β-cellsretain the capacity to regenerate through proliferation. Accumulatingevidence suggests that even in humans the β-cell maintains itsproliferative capacity. However, the normal rate at which human adultβ-cells replicate is very slow. Therefore, one of the top priorities inthe treatment of diabetes is to identify agents that have the potentialto enhance human β-cell regeneration in vivo.

It is documented herein that OPG can stimulate endogenous β-cellreplication in rodents in vivo, and more importantly, human β-cellproliferation in vitro. The studies disclosed herein clearly show thatOPG can induce human β-cell proliferation. Besides its proliferativeeffects, OPG also has pro-survival effects on the β-cell. OPG has thepotential to expand, regenerate, and preserve endogenous humanfunctional β-cell mass in vivo under basal and stress-induced conditionsand has the capacity to induce proliferation, survival and function inhuman β-cells.

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

α cells: Mature glucagon producing cells. In vivo, these cells are foundin the pancreatic islets of Langerhans.

β cells: Mature insulin producing cells. In vivo, these cells are foundin the pancreatic islets of Langerhans,

δ cells: Mature somatostatin producing cells. In vivo, these cells arefound in the pancreatic islets of Langerhans.

PP cells: Mature pancreatic polypeptide (PP) producing cells. In vivo,these cells are found in the pancreatic islets of Langerhans.

ε cells: Mature ghrelin producing cells. In vivo, these cells are foundin the pancreatic islets of Langerhans.

Administration: To provide or give a subject an agent by any effectiveroute. Exemplary routes of administration include, but are not limitedto, oral, injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, intravenous, and intratumoral), sublingual, rectal,transdermal, intranasal, vaginal and inhalation routes.

Agent: Any polypeptide, compound, small molecule, organic compound,salt, polynucleotide, or other molecule of interest. Agent can include atherapeutic agent, a diagnostic agent or a pharmaceutical agent. Atherapeutic agent is a substance that demonstrates some therapeuticeffect by restoring or maintaining health, such as by alleviating thesymptoms associated with a disease or physiological disorder, ordelaying (including preventing) progression or onset of a disease.

Amplification: Of a nucleic acid molecule (such as, a DNA or RNAmolecule) refers to use of a technique that increases the number ofcopies of a nucleic acid molecule in a specimen. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of amplification maybe characterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Anti-diabetic lifestyle modifications: Changes to lifestyle, habits, andpractices intended to alleviate the symptoms of diabetes orpre-diabetes. Obesity and sedentary lifestyle may both independentlyincrease the risk of a subject developing type II diabetes, soanti-diabetic lifestyle modifications include those changes that willlead to a reduction in a subject's body mass index (BMI), increasephysical activity, or both. Specific, non-limiting examples include thelifestyle interventions described in Diabetes Care, 22(4):623-34 atpages 626-27, herein incorporated by reference.

Artificial Islets: Clusters of pancreatic endocrine cells formed by thedifferentiation of stem or progenitor cells including ES cell in vitro,dislodged clusters of pancreatic endocrine cells, endocrine cellsdifferentiated from stem cells or progenitor cells including ES cells invitro, cells that have undergone a mesenchymal-to-epithelial orepithelial-to-mesenchymal-to-epithelial transition or endocrine cellsaggregated into a cluster in vitro.

Diabetes mellitus: A group of metabolic diseases in which a subject hashigh blood sugar, either because the pancreas does not produce enoughinsulin, or because cells do not respond to the insulin that isproduced. Type 1 diabetes results from the body's failure to produceinsulin. This form has also been called “insulin-dependent diabetesmellitus” (IDDM) or “juvenile diabetes”. Type 1 diabetes mellitus ischaracterized by loss of the insulin-producing βcells, leading toinsulin deficiency. This type can be further classified asimmune-mediated or idiopathic. Type 2 diabetes results from insulinresistance, a condition in which cells fail to use insulin properly,sometimes combined with an absolute insulin deficiency. This form isalso called “non insulin-dependent diabetes mellitus” (NIDDM) or“adult-onset diabetes.” The defective responsiveness of body tissues toinsulin is believed to involve the insulin receptor. Diabetes mellitusis characterized by recurrent or persistent hyperglycemia, and isdiagnosed by demonstrating any one of:

-   -   a. Fasting plasma glucose level≧7.0 mmol/l (126 mg/dl);    -   b. Plasma glucose≧11.1 mmol/l (200 mg/dL) two hours after a 75 g        oral glucose load as in a glucose tolerance test;    -   c. Symptoms of hyperglycemia and casual plasma glucose≧11.1        mmol/l (200 mg/dl);    -   d. Glycated hemoglobin (Hb A1C)≧6.5%

Differentiation: The process whereby relatively unspecialized cells(e.g., embryonic cells) acquire specialized structural and/or functionalfeatures characteristic of mature cells. Similarly, “differentiate”refers to this process. Typically, during differentiation, cellularstructure alters and tissue-specific proteins appear. The term“differentiated pancreatic endocrine cell” refers to cells expressing aprotein characteristic of the specific pancreatic endocrine cell type. Adifferentiated pancreatic endocrine cell includes an α cell, a β cell, aδ cell, and a PP cell, which express glucagon, insulin, somatostatin,and pancreatic polypeptide, respectively.

Growth factor: A substance that promotes cell growth, survival, and/ordifferentiation. Growth factors include molecules that function asgrowth stimulators (mitogens), molecules that function as growthinhibitors (e.g. negative growth factors) factors that stimulate cellmigration, factors that function as chemotactic agents or inhibit cellmigration or invasion of tumor cells, factors that modulatedifferentiated functions of cells, factors involved in apoptosis, orfactors that promote survival of cells without influencing growth anddifferentiation. Examples of growth factors are fibroblast growth factor(FGF)2, epidermal growth factor (EGF), ciliary neurotrophic factor(CNTF), hepatocyte growth factor (HGF), nerve growth factor (NGF), andactvin-A.

Effective amount or Therapeutically effective amount: The amount ofagent, such as OPG, a functional fragment or variant thereof, that is anamount sufficient to prevent, treat (including prophylaxis), reduceand/or ameliorate the symptoms and/or underlying causes of any of adisorder or disease. In one embodiment, an “effective amount” issufficient to reduce or eliminate a symptom of a disease, such as apancreatic cancer. In another embodiment, an effective amount is anamount sufficient to overcome the disease itself.

Endocrine: Tissue which secretes regulatory hormones directly into thebloodstream without the need for an associated duct system.

Expand: A process by which the number or amount of cells in a cellculture is increased due to cell division. Similarly, the terms“expansion” or “expanded” refers to this process. The terms“proliferate,” “proliferation” or “proliferated” may be usedinterchangeably with the words “expand,” “expansion,” or “expanded.”Typically, during an expansion phase, the cells do not differentiate toform mature cells.

Expressed: Translation of a nucleic acid into a protein. Proteins may beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Exocrine: Secretory tissue which distributes its products, such asenzymes, via an associated duct network. The exocrine pancreas is thepart of the pancreas that secretes enzymes required for digestion. Theexocrine cells of the pancreas include the centroacinar cells andbasophilic cells, which produce secretin and cholecystokinin.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Heterologous: A heterologous sequence is a sequence that is not normally(in the wild-type sequence) found adjacent to a second sequence. In oneembodiment, the sequence is from a different genetic source, such as avirus or organism, than the second sequence.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Islets of Langerhans: Small discrete clusters of pancreatic endocrinetissue. In vivo, in an adult mammal, the islets of Langerhans are foundin the pancreas as discrete clusters (islands) of pancreatic endocrinetissue surrounded by the pancreatic exocrine (or acinar) tissue. Invivo, the islets of Langerhans consist of the α cells, β cells, δ cells,PP cells, and ε cells. Histologically, in rodents, the islets ofLangerhans consist of a central core of β cells surrounded by an outerlayer of α cells, δ cells, and PP cells. The structure of human isletsof Langerhans is different and distinct from rodents. The islets ofLangerhans are sometimes referred to herein as “islets.”

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids. An isolated cell type has been substantially separatedfrom other cell types, such as a different cell type that occurs in anorgan. A purified cell or component can be at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% pure.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, such as a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (such as a promoter, origin ofreplication, ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, for example, by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, bythe homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see for example, Current Protocols in Molecular Biology(Ausubel et al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, such as version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. The BLASTNprogram (for nucleotide sequences) uses as defaults a word length (W) of11, alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands. The BLASTP program (for amino acidsequences) uses as defaults a word length (W) of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915, 1989).

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Osteoprotegerin (OPG): A protein of the Tumor Necrosis Factor (TNF)receptor family, also known as osteoclastogenesis inhibitory factor(OCIF), or tumor necrosis factor receptor superfamily member 11B(TNFRSF11B), was first described by Simonet et al. (Cell, 89, 309-319(1997)). OPG appears to be a crucial element in regulating the naturalprocesses of bone production and turnover. Changes in the balancebetween OPG and its target receptor activator of nuclear factor kappa Bligand (RANKL) have been noted in a number of conditions associated withabnormal bone metabolism. OPG has undergone preclinical and clinicaltesting for conditions associated with increased bone turnover and boneloss, including osteoporosis, rheumatoid arthritis, Paget's disease,periodontal disease, vascular disease and cancers that are located in orhave metastasized to bone (For review, see Lorenz et al., J. Amer MedAssoc 292: 490-5 (2004)). OPG is disclosed in U.S. Pat. No. 6,015,938,U.S. Pat. No. 6,284,740, U.S. Pat. No. 6,284,728, U.S. Pat. No.6,613,544, U.S. Pat. No. 6,316,408, U.S. Pat. No. 6,288,032 and U.S.Pat. No. 6,369,027, which are incorporated herein by reference.

Pancreatic endocrine cell: An endocrine cell of pancreatic origin thatproduces one or more pancreatic hormone, such as insulin, glucagon,somatostatin, or pancreatic polypeptide. Subsets of pancreatic endocrinecells include the α (glucagon producing), β (insulin producing) δ(somatostatin producing) or PP (pancreatic polypeptide producing) cells.Additional subsets produce more than one pancreatic hormone, such as,but not limited to, a cell that produces both insulin and glucagon, or acell that produces insulin, glucagon, and somatostatin, or a cell thatproduces insulin and somatostatin.

Pancreatic cancer: A malignant tumor within the pancreas. The prognosisis generally poor. About 95% of pancreatic cancers are adenocarcinomas.The remaining 5% are tumors of the exocrine pancreas (for example,serous cystadenomas), acinar cell cancers, and pancreatic neuroendocrinetumors (such as insulinomas). An “insulinoma” is a cancer of the betacells that retains the ability to secrete insulin. Patients withinsulinomas usually develop neuroglycopenic symptoms. These includerecurrent headache, lethargy, diplopia, and blurred vision, particularlywith exercise or fasting. Severe hypoglycemia may result in seizures,coma and permanent neurological damage. Symptoms resulting from thecatecholaminergic response to hypoglycemia (for example, tremulousness,palpitations, tachycardia, sweating, hunger, anxiety, nausea). Apancreatic adenocarciona occurs in the glandular tissue. Symptomsinclude abdominal pain, loss of appetite, weight loss, jaundice andpainless extension of the gallbladder.

Classical treatment for pancreatic cancer, including adenocarcinomas andinsulinomas includes surgical resection (such as the Whipple procedure)and chemotherapy with agent such as fluorouracil, gemcitabine, anderlotinib.

Pre-diabetes: A state in which some, but not all, of the criteria fordiabetes are met. For example, a subject can have impaired fastingglycaemia or impaired fasting glucose (IFG). Subjects with fastingglucose levels from 110 to 125 mg/dl (6.1 to 6.9 mmol/1) are consideredto have impaired fasting glucose. Subjects with plasma glucose at orabove 140 mg/dL (7.8 mmol/L), but not over 200 mg/dL (11.1 mmol/L), twohours after a 75 g oral glucose load are considered to have impairedglucose tolerance.

Predisposition for diabetes: A subject that is at high risk fordeveloping diabetes. A number of risk factors are known to those ofskill in the art and include: genetic factors (e.g., carrying allelesthat result in a higher occurrence of diabetes than in the averagepopulation or having parents or siblings with diabetes); overweight(e.g., body mass index (BMI) greater or equal to 25 kg/m.sup.2);habitual physical inactivity, race/ethnicity (e.g., African-American,Hispanic-American, Native Americans, Asian-Americans, PacificIslanders); previously identified impaired fasting glucose or impairedglucose tolerance, hypertension (e.g., greater or equal to 140/90 mmHgin adults); HDL cholesterol greater or equal to 35 mg/dl; triglyceridelevels greater or equal to 250 mg/dl; a history of gestational diabetesor delivery of a baby over nine pounds; and/or polycystic ovarysyndrome. See, e.g., “Report of the Expert Committee on the Diagnosisand Classification of Diabetes Mellitus” and “Screening for Diabetes”Diabetes Care 25(1): S5-S24 (2002).

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide” or“protein” as used herein is intended to encompass any amino acidsequence and include modified sequences such as glycoproteins. The term“polypeptide” is specifically intended to cover naturally occurringproteins, as well as those that are recombinantly or syntheticallyproduced.

The term “polypeptide fragment” refers to a portion of a polypeptidewhich exhibits at least one useful epitope. The term “functionalfragments of a polypeptide” refers to all fragments of a polypeptidethat retain an activity of the polypeptide. Biologically functionalfragments, for example, can vary in size from a polypeptide fragment assmall as an epitope capable of binding an antibody molecule to a largepolypeptide capable of participating in the characteristic induction orprogramming of phenotypic changes within a cell. An “epitope” is aregion of a polypeptide capable of binding an immunoglobulin generatedin response to contact with an antigen. Thus, smaller peptidescontaining the biological activity of insulin, or conservative variantsof the insulin, are thus included as being of use.

The term “soluble” refers to a form of a polypeptide that is notinserted into a cell membrane.

The term “substantially purified polypeptide” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Variations in thecDNA sequence that result in amino acid changes, whether conservative ornot, should be minimized in order to preserve the functional andimmunologic identity of the encoded protein. The immunologic identity ofthe protein may be assessed by determining if it is recognized by anantibody; a variant that is recognized by such an antibody isimmunologically conserved. Any cDNA sequence variant will preferablyintroduce no more than twenty, and preferably fewer than ten amino acidsubstitutions into the encoded polypeptide. Variant amino acid sequencesmay, for example, be 80, 90 or even 95% or 98% identical to the nativeamino acid sequence.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or a composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for a drug to interact with a cell. “Contacting” includesincubating a drug in solid or in liquid form with a cell.

Polynucleotide: A nucleic acid sequence (such as a linear sequence) ofany length. Therefore, a polynucleotide includes oligonucleotides, andalso gene sequences found in chromosomes. An “oligonucleotide” is aplurality of joined nucleotides joined by native phosphodiester bonds.An oligonucleotide is a polynucleotide of between 6 and 300 nucleotidesin length. An oligonucleotide analog refers to moieties that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA, and includepeptide nucleic acid (PNA) molecules.

Primers: Short nucleic acids, for example DNA oligonucleotides 10nucleotides or more in length, which are annealed to a complementarytarget DNA strand by nucleic acid hybridization to form a hybrid betweenthe primer and the target DNA strand, then extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, such as by the polymerasechain reaction (PCR) or other nucleic-acid amplification methods knownin the art.

Probes and primers as used in the present invention may, for example,include at least 10 nucleotides of the nucleic acid sequences that areshown to encode specific proteins. In order to enhance specificity,longer probes and primers may also be employed, such as probes andprimers that comprise 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100consecutive nucleotides of the disclosed nucleic acid sequences. Methodsfor preparing and using probes and primers are described in thereferences, for example Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, New York; Ausubel et al. (1987)Current Protocols in Molecular Biology, Greene Publ. Assoc. &Wiley-Intersciences; Innis et al. (1990) PCR Protocols, A Guide toMethods and Applications, Innis et al. (Eds.), Academic Press, SanDiego, Calif. PCR primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

When referring to a probe or primer, the term “specific” for (a targetsequence) indicates that the probe or primer hybridizes under stringentconditions substantially only to the target sequence in a given samplecomprising the target sequence.

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, such as by genetic engineering techniques. Similarly,a recombinant protein is one encoded for by a recombinant nucleic acidmolecule.

Sequence identity of amino acid sequences: The similarity between aminoacid sequences is expressed in terms of the similarity between thesequences, otherwise referred to as sequence identity. Sequence identityis frequently measured in terms of percentage identity (or similarity orhomology); the higher the percentage, the more similar the two sequencesare. Homologs or variants of a polypeptide will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of OPG are typically characterized by possessionof at least about 75%, for example at least about 80%, 90%, 95%, 96%,97%, 98% or 99% sequence identity counted over the full length alignmentwith the amino acid sequence of the antibody using the NCBI Blast 2.0,gapped blastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a β cell specific binding agent is an agent thatbinds substantially to a β cell, and a pancreatic endocrine cellspecific binding agent is an gent that binds substantially only topancreatic endocrine cells or a subset thereof (and not to pancreaticexocrine cells). Similarly, a pancreatic exocrine cell specific bindingagent is an agent that binds substantially to exocrine cells. In oneembodiment, the specific binding agent is a monoclonal or polyclonalantibody that specifically binds a type of pancreatic cell.

The term “specifically binds” refers, with respect to a cell, such as apancreatic endocrine cell, to the preferential association of anantibody or other ligand, in whole or part, with a cell or tissuebearing that antigen and not to cells or tissues lacking that antigen.It is, of course, recognized that a certain degree of non-specificinteraction may occur between a molecule and a non-target cell ortissue. Nevertheless, specific binding may be distinguished as mediatedthrough specific recognition of the antigen. Although selectivelyreactive antibodies bind antigen, they may do so with low affinity. Onthe other hand, specific binding results in a much stronger associationbetween the antibody (or other ligand) and cells bearing the antigenthan between the bound antibody (or other ligand) and cells lacking theantigen. Specific binding typically results in greater than 2-fold, suchas greater than 5-fold, greater than 10-fold, or greater than 100-foldincrease in amount of bound antibody or other ligand (per unit time) toa cell or tissue expressing the target epitope as compared to a cell ortissue lacking this epitope. Specific binding to a protein under suchconditions requires an antibody that is selected for its specificity fora particular protein. A variety of immunoassay formats are appropriatefor selecting antibodies or other ligands specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow & Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

Subject: Any mammal, such as humans, non-human primates, pigs, sheep,cows, rodents and the like which is to be the recipient of theparticular treatment. In two non-limiting examples, a subject is a humansubject or a murine subject.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents. A therapeutic agent can bean antibody that specifically binds pancreatic endocrine cells or asubset thereof.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” or“transfected” by a nucleic acid transduced into the cell when the DNAbecomes stably replicated by the cell, either by incorporation of thenucleic acid into the cellular genome, or by episomal replication.

Numerous methods of transfection are known to those skilled in the art,such as: chemical methods (e.g., calcium-phosphate transfection),physical methods (e.g., electroporation, microinjection, particlebombardment), fusion (e.g., liposomes), receptor-mediated endocytosis(e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes) andby biological infection by viruses such as recombinant viruses {Wolff,J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)}. In thecase of infection by retroviruses, the infecting retrovirus particlesare absorbed by the target cells, resulting in reverse transcription ofthe retroviral RNA genome and integration of the resulting provirus intothe cellular DNA. Methods for the introduction of genes into thepancreatic endocrine cells are known (e.g. see U.S. Pat. No. 6,110,743,herein incorporated by reference). These methods can be used totransduce a pancreatic endocrine cell produced by the methods describedherein, or an artificial islet produced by the methods described herein.

Genetic modification of the target cell is an indicium of successfultransfection. “Genetically modified cells” refers to cells whosegenotypes have been altered as a result of cellular uptakes of exogenousnucleotide sequence by transfection. A reference to a transfected cellor a genetically modified cell includes both the particular cell intowhich a vector or polynucleotide is introduced and progeny of that cell.

Transgene: An exogenous gene supplied by a vector.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more therapeuticgenes and/or selectable marker genes and other genetic elements known inthe art. A vector can transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. A vector optionally includes materials to aidin achieving entry of the nucleic acid into the cell, such as a viralparticle, liposome, protein coating or the like.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Osteoprotegerin (OPG) and Variants Thereof

OPG is a member of the TNF receptor superfamily Human wild-type OPG hasthe amino acid sequence set forth as

MNKLLCCALV FLDISIKWTT QETFPPKYLH YDEETSHQLLCDKCPPGTYL KQHCTAKWKT VCAPCPDHYY TDSWHTSDECLYCSPVCKEL QYVKQECNRT HNRVCECKEG RYLEIEFCLKHRSCPPGFGV VQAGTPERNT VCKRCPDGFF SNETSSKAPCRKHTNCSVFG LLLTQKGNAT HDNICSGNSE STQKCGIDVTLCEEAFFRFA VPTKFTPNWL SVLVDNLPGT KVNAESVERIKRQHSSQEQT FQLLKLWKHQ NKAQDIVKKI IQDIDLCENSVQRHIGHANL TFEQLRSLME SLPGKKVGAE DIEKTIKACKPSDQILKLLS LWRIKNGDQD TLKGLMHALK HSKTYHFPKTVTQSLKKTIR FLHSFTMYKL YQKLFLEMIG NQVQSVKISC L (GENBANK ®Accession No. AAB53709.1 and NP_002537.3, both incorporated herein byreference, SEQ ID NO: 1).

Wild-type OPG is an atypical member of the TNF receptor superfamily(TNFRS), in that it is a secreted protein with no transmembrane domainand no direct signaling properties. OPG secretion is through a21-residue signal peptide which is cleaved, generating a mature proteinof 380 amino acids. Thus, in some embodiments, the method includesadministering an OPG functional fragment including or consisting ofamino acids 22-401 of SEQ ID NO: 1. OPG sequences of use encompass thosethat have the amino terminal leader sequence of 21 amino acids removedand/or where amino acids are removed from the C-terminus up to andincluding amino acid 185.

OPG has three major structural motifs: i) four cysteine-rich TNFreceptor domains which are necessary and sufficient for binding to itstarget; ii) a heparin-binding domain that is involved in homodimerformation; and iii) two death domain homologous (DDH) regions withunknown functional role (FIG. 1). Wild-type OPG is produced as a monomer(55-62 kDa), but gets dimerized and is secreted as a disulfide-linkedhomodimeric glycoprotein of 110-120 kDa.

A variant of OPG can be at least 95%, 96%, 97%, 98%, 99% identical toSEQ ID NO: 1, or amino acids 22-410 of SEQ ID NO: 1; these variants areof use in the disclosed methods. In some embodiments, these variantsretain the ability to form dimers and interact with RANKL and/or bindTNF-related apoptosis inducing ligand (TRAIL). OPG is a decoy receptorfor TRAIL, modulates TRAIL's ability to target cells. Without beingbound by theory, OPG is active in bone metabolism, and particular itinhibits bone resorption and increases bone density. The interaction ofOPG with RANKL inhibits RANKL's ability to stimulate the formation andactivity of osteoclasts via its interaction with RANK, and it is thisactivity of OPG that confers its ability to reduce bone loss. Thebiological activities of OPG also include activity associated withbinding to TRAIL.

Active functional fragments of OPG can also be used in the methodsdisclosed herein. Yamaguchi et al. (J. Biol. Chem. 273, 5117-5123, 1998,incorporated herein by reference) demonstrated that truncated anddeleted variants of OPG retained OPG-like activity, while OPG in bothmonomer or dimer form retained biological activity (Tomoyasu, A et al.,Biochem Biophys Res Commun 245, 382-387 (1998)). Schneeweis et al., (J.Biol. Chem. Oct. 7, 2005 (epub)) examined the assembly, state andaffinity of OPG for RANKL. Dimerization of OPG either in full lengthform, where the dimerization is mediated by non-covalent interactionswithin the death domain regions, or in truncated form, such as wheredimerization may be mediated by Fc attachment, results in high affinityattachment of OPG to RANKL.

Variants of OPG that retain binding to RANKL are also known, see U.S.Published Patent Application No. 2010/0144600 and U.S. Pat. No.7,612,169, both incorporated herein by reference. In some embodiments,an OPG variant protein is a modified version of a full length wild typeOPG sequence. In another embodiment, the OPG variant protein containsone or more additional insertions or deletions at the N-terminus,C-terminus, or internally. For example, the OPG variant protein mayconsist of any modified active functional fragment of OPG. The activefunctional fragment may consist of, for example, amino acids 22-294,amino acids 22-201, 22-194 or amino acids 1-197.

In some embodiments, OPG variant proteins have at least one amino acidresidue that differs from a wild-type OPG sequence, such as at most 2,3, 4, or 5 different residues. The modifications can be amino acidsubstitutions and may include those to surface or exposed areas of OPG.OPG variant proteins may include one domain or multiple domainsconnected by linker sequences. OPG variant proteins can contain furthermodifications, for instance modifications that alter stability orimmunogenicity or which enable posttranslational modifications such asPEGylation or glycosylation.

In one embodiment, the OPG variant protein includes at least onemodification in the region encompassed by amino acid residues 102-130.For example, the OPG variant protein includes at least one amino acidsubstitution at positions 102, 111, 115, 122, 128 or 130. In otherembodiments, the OPG variant protein includes at least one modificationwithin the loop structure comprising residues 107-118. In anotherembodiment, the modification in this region includes substitution of Ileat position 115. Ile at position 115 can be substituted by amino acidsthat are more polar or have shorter side chains. In some examples, Ileat position 115 is substituted with Thr, Met, Val, Asp, Gly. Ser or Arg.Thus, modifications of use include I115T, I115M, I115V, I115D, I115G,I115S and I115R.

In some embodiments, the OPG variant protein comprises at least onemodification in the region encompassed by amino acid residues 120-130.In another embodiment, the modification involves substitution of Arg atposition 122. In some examples, Arg at position 122 is substituted withGly, Gln, Ser, Asn or Glu. Thus, modifications include R122G, R122Q,R122S, R122N and R122E. In other examples, the modification involvessubstitution of Phe at position 128. For example, Phe at position 128can be substituted with Val, Ala, Leu, Ile or Ser. Thus, modificationsinclude F128V, F128A, F128L, F128 I and F128S. In other examples, themodification involves substitution of Val at position 130. For example,Val at position 130 is substituted with Glu or Ala.

In some embodiments, the OPG variant protein comprises at least twomodifications or at most two modifications. The at two modifications canboth occur in the region encompassed by residues 102-130. For example,at least two modifications can include substitutions at positions 115and 122. Examples of suitable double modifications within this regionare R122N and I115M; R122N and I115M; F128S and I115M; F128I and I115M;and F128L and I115M. Alternatively, at least two modifications caninclude one or more modifications within the region encompassed by aminoacids 102-130 and one or more modifications outside of this region. Themodification outside of this region can occur at, for example, any oneor more of residues 31, 40, 51, 100, 155, 167 or 168. In one embodiment,the residue 40 is modified. In some embodiments, Leu at position 40 issubstitute with Ser.

In another embodiment, the OPG variant protein of use in the presentmethods comprises one or more modifications within the regionencompassed by amino acids 102-130 and a modification to any one or moreof the following amino acid residues: Gln21, Glu22, Thr23, Phe24, Pro25,Pro26, Lys27, Tyr28, Leu29, His30, Tyr31, Asp32, Glu33, Glu34, Thr35,Ser36, His37, Gln38, Asp42, Lys43, Pro45, Pro46, Thr48, Lys51, Gln52,His53, Cys54, Thr55, Ala56, Lys57, Trp58, Lys59, Thr60, Val61, Ala63,Pro64, Pro66, Asp67, His68, Tyr69, Asp72, Ser73, Trp74, Thr76, Ser77,Asp78, Glu79, Leu81, Tyr82, Ser84, Pro85, Val86, Lys88, Glu89, Leu90,Tyr92, Val93, Lys94, Gln95, Glu96, Asn98, Arg99, Thr100, His101, Val131,Gln132, Ala133, Gly134, Thr135, Pro136, Glu137, Arg138, Val141, Lys143,Arg144, Cys145, Pro146, Asp147, Gly148, Phe149, Phe150, Ser151, Asn152,Glu153, Thr154, Ser155, Ser156, Lys157, Ala158, Pro159, Cys160, Arg161,Lys162, His163, Thr164, Asn165, Cys166, Ser167, Val168, Phe169, Gly170,Leu171, Leu172, Leu173, Thr174, Gln175, Lys176, Gly177, Asn178, Ala179,Thr180, His181, Asp182, Asn183, Ile184, Cys185, Ser186, Gly187, Asn188,Ser189, Glu190, Ser191, Thr192, Gln193, Lys194, Cys195, Gly196, Ile197,Asp198, Val199, Thr200 and Leu201. In other embodiments the OPG variantprotein of use in the present methods includes one or more modificationswithin the region encompassed by amino acids 102-130 and a modificationto any one or more of the following amino acid residues: Tyr28, His30,Tyr31, Glu34, Thr35, Ser36, His 37, Lys43, Tyr49, Gln52, His53, Pro66,Asp67, His68, Tyr69, Tyr70, Thr71, Asp72, Ser73, Trp74, His75, Thr76,Ser77, Asp78, Glu79, Cys80, Leu81, Tyr82, Cys83, Ser84, Pro85, Val86,Cys87, Lys88, Glu89, Leu90, Gln91, Asn139 and Glu153. Additionalvariants of OPG of use are disclosed in U.S. Published PatentApplication No. 2010/0144600, which is incorporated herein by reference.

OPG can be included in a fusion protein. Thus, in some embodiments, theOPG is administered as a fusion protein, such as an Fc fusion protein.In some specific, non-liming examples, the Fc domain is an IgG Fcdomain, such as an IgG₁, IgG₂, IgG₃ or an IgG₄ Fc domain. In someembodiments, these forms of OPG have an increased half-life as comparedto the OPG not included in the fusion protein. Exemplary fusion proteinsare commercially available, such as Human Osteoprotegerin/TNFRSF11B FcChimera from R and D Systems, Catalog Number: 805-OS and MouseOsteoprotegerin/TNFRSF11B Fc Chimera from R and D Systens, CatalogNumber 459-MO.

Without being bound by theory, the Fc domain increases the half-life ofan IgG through its unique pH-dependent association with the neonatal Fcreceptor (FcRn). After internalization, the Fc domain of IgG can bind toFcRn in the acidic environment of the endosome, so that the IgG is thencycled onto the cell surface and re-released into circulation. Thisbiological system protects IgG from degradation and results in a longserum half-life. Fusions of an Fc domain and a therapeutic molecule havean extended half life. In addition, since the Fc fragment of IgGconsists of a tightly packed homodimer, two therapeutic proteins arepresent in each molecule. Recently, monomeric Fc fusion proteins weregenerated in which a single active protein was fused to dimericwild-type Fc. These smaller molecules have been shown to possess evenextended half-lives compared with the dimeric version.

Polynucleotides and Vectors

Polynucleotide molecules encoding OPG, a functional fragment, variant,or fusion protein thereof can readily be produced by one of skill in theart, using the amino acid sequences provided herein, and the geneticcode. In addition, one of skill can readily construct a variety ofclones containing functionally equivalent nucleic acids, such as nucleicacids which differ in sequence but which encode protein sequence. AnExemplary nucleic acid sequence encoding human OPG is provided below.

(SEQ ID NO: 2) GTATATATAA CGTGATGAGC GTACGGGTGC GGAGACGCACCGGAGCGCTC GCCCAGCCGC CGYCTCCAAG CCCCTGAGGTTTCCGGGGAC CACAATGAAC AAGTTGCTGT GCTGCGCGCTGACATCTCCA TTAAGTGGAC CACCCAGGAA ACGTTTCCTCCAAAGTACCT TCATTATGAC GAAGAAACCT CTCATCAGCTGTTGTGTGAC AAATGTCCTC CTGGTACCTA CCTAAAACAACACTGTACAG CAAAGTGGAA GACCGTGTGC GCCCCTTGCCCTGACCACTA CTACACAGAC AGCTGGCACA CCAGTGACGAGTGTCTATAC TGCAGCCCCG TGTGCAAGGA GCTGCAGTACGTCAAGCAGG AGTGCAATCG CACCCACAAC CGCGTGTGCGAATGCAAGGA AGGGCGCTAC CTTGAGATAG AGTTCTGCTTGAAACATAGG AGCTGCCCTC CTGGATTTGG AGTGGTGCAAGCTGGAACCC CAGAGCGAAA TACAGTTTGC AAAAGATGTCCAGATGGGTT CTTCTCAAAT GAGACGTCAT CTAAAGCACCCTGTAGAAAA CACACAAATT GCAGTGTCTT TGGTCTCCTGCTAACTCAGA AAGGAAATGC AACACACGAC AACATATGTTCCGGAAACAG TGAATCAACT CAAAAATGTG GAATAGATGTTACCCTGTGT GAGGAGGCAT TCTTCAGGTT TGCTGTTCCTACAAAGTTTA CGCCTAACTG GCTTAGTGTC TTGGTAGACAATTTGCCTGG CACCAAAGTA AACGCAGAGA GTGTAGAGAGGATAAAACGG CAACACAGCT CACAAGAACA GACTTTCCAGCTGCTGAAGT TATGGAAACA TCAAAACAAA GCCCAAGATATAGTCAAGAA GATCATCCAA GATATTGACC TCTGTGAAAACAGCGTGCAG CGGCACATTG GACATGCTAA CCTCACCTTCGAGCAGCTTC GTAGCTTGAT GGAAAGCTTA CCGGGAAAGAAAGTGGGAGC AGAAGACATT GAAAAAACAA TAAAGGCATGCAAACCCAGT GACCAGATCC TGAAGCTGCT CAGTTTGTGGCGAATAAAAA ATGGCGACCA AGACACCTTG AAGGGCCTAATGCACGCACT AAAGCACTCA AAGACGTACC ACTTTCCCAAAACTGTCACT CAGAGTCTAA AGAAGACCAT CAGGTTCCTTCACAGCTTCA CAATGTACAA ATTGTATCAG AAGTTATTTTTAGAAATGAT AGGTAACCAG GTCCAATCAG TAAAAATAAGCTGCTTATAA CTGGAAATGG CCATTGAGCT GTTTCCTCAC AATTGGCGAG ATCCCATGGA TGATAAPolynucleotides include DNA, cDNA and RNA sequences which encode thepeptide of interest. Silent mutations in the coding sequence result fromthe degeneracy (i.e., redundancy) of the genetic code, whereby more thanone codon can encode the same amino acid residue. Thus, for example,leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can beencoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded byAAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can beencoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG;glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TATor TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tablesshowing the standard genetic code can be found in various sources (see,for example, Stryer, 1988, Biochemistry, 3.sup.rd Edition, W.H. 5Freeman and Co., NY).

A nucleic acid encoding OPG, variants thereof and fusion proteins can becloned or amplified by in vitro methods, such as the polymerase chainreaction (PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (3SR) and the Qβ replicase amplification system (QB). Forexample, a polynucleotide encoding the protein can be isolated bypolymerase chain reaction of cDNA using primers based on the DNAsequence of the molecule. A wide variety of cloning and in vitroamplification methodologies are well known to persons skilled in theart. PCR methods are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; andErlich, ed., PCR Technology, (Stockton Press, N Y, 1989).Polynucleotides also can be isolated by screening genomic or cDNAlibraries with probes selected from the sequences of the desiredpolynucleotide under stringent hybridization conditions.

Nucleic acid sequences encoding OPG, a functional fragment, variant, orfusion protein thereof can be prepared by any suitable method including,for example, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids encoding sequences encoding OPG, a functionalfragment, variant, or fusion protein thereof can be prepared by cloningtechniques. Examples of appropriate cloning and sequencing techniques,and instructions sufficient to direct persons of skill through cloningare found in Sambrook et al., supra, Berger and Kimmel (eds.), supra,and Ausubel, supra. Product information from manufacturers of biologicalreagents and experimental equipment also provide useful information.Such manufacturers include the SIGMA® Chemical Company (Saint Louis,Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway,N.J.), CLONTECH® Laboratories, Inc. (Palo Alto, Calif.), Chem GenesCorp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,GIBCO® BRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),INVITROGEN® (San Diego, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methodsAmplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

In one example, OPG, a functional fragment, variant, or fusion proteinthereof is prepared by inserting the cDNA which encodes the protein intoa vector. The insertion can be made so that the protein and aheterologous protein are read in frame so that one continuouspolypeptide is produced.

Once the nucleic acids encoding OPG, a functional fragment, variant, orfusion protein thereof are isolated and cloned, the protein can beexpressed in a recombinantly engineered cell such as bacteria, plant,yeast, insect and mammalian cells using a suitable expression vector.One or more DNA sequences OPG, a functional fragment, variant, or fusionprotein thereof can be expressed in vitro by DNA transfer into asuitable host cell. The cell may be prokaryotic or eukaryotic. The termalso includes any progeny of the subject host cell. It is understoodthat all progeny may not be identical to the parental cell since theremay be mutations that occur during replication. Methods of stabletransfer, meaning that the foreign DNA is continuously maintained in thehost, are known in the art.

Polynucleotide sequences encoding OPG, a functional fragment, variant,or fusion protein thereof can be operatively linked to expressioncontrol sequences. An expression control sequence operatively linked toa coding sequence is ligated such that expression of the coding sequenceis achieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding OPG, a functional fragment,variant, or fusion protein thereof can be inserted into an expressionvector including, but not limited to a plasmid, virus or other vehiclethat can be manipulated to allow insertion or incorporation of sequencesand can be expressed in either prokaryotes or eukaryotes. Hosts caninclude microbial, yeast, insect and mammalian organisms. Methods ofexpressing DNA sequences having eukaryotic or viral sequences inprokaryotes are well known in the art. Biologically functional viral andplasmid DNA vectors capable of expression and replication in a host areknown in the art.

In one embodiment, vectors are used for expression in yeast such as S.cerevisiae or Kluyveromyces lactis. Several promoters are known to be ofuse in yeast expression systems such as the constitutive promotersplasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). Inaddition, many inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHO5 (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive MET3 and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (2μ) or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (AMP) for propagation inbacteria. Plasmids for expression on K. lactis are known, such aspKLAC1. Thus, in one example, after amplification in bacteria, plasmidscan be introduced into the corresponding yeast auxotrophs by methodssimilar to bacterial transformation. The polynucleotides can also bedesigned to express in insect cells.

OPG, a functional fragment, variant, or fusion protein thereof can beexpressed in a variety of yeast strains. For example, seven pleiotropicdrug-resistant transporters, YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, andPDR15, together with their activating transcription factors, PDR1 andPDR3, have been simultaneously deleted in yeast host cells, renderingthe resultant strain sensitive to drugs. Yeast strains with alteredlipid composition of the plasma membrane, such as the erg6 mutantdefective in ergosterol biosynthesis, can also be utilized. Proteinsthat are highly sensitive to proteolysis can be expressed in a yeastlacking the master vacuolar endopeptidase Pep4, which controls theactivation of other vacuolar hydrolases. Heterologous expression instrains carrying temperature-sensitive (ts) alleles of genes can beemployed if the corresponding null mutant is inviable.

Viral vectors can also be prepared encoding OPG, a functional fragment,variant, or fusion protein thereof. A number of viral vectors have beenconstructed, including polyoma, SV40 (Madzak et al., 1992, J. Gen.Virol., 73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol.Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629;Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992,Proc. Nad. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell,68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses of avian(Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouploset al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top.Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol.,5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann etal., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990,J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol.,66:2731-2739). Baculovirus (Autographa californica multinuclearpolyhedrosis virus; AcMNPV) vectors are also known in the art, and maybe obtained from commercial sources (such as PharMingen, San Diego,Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla,Calif.). Thus, in one embodiment, the polynucleotide encoding OPG, afunctional fragment, variant, or fusion protein thereof is included in aviral vector. Suitable vectors include retrovirus vectors, orthopoxvectors, avipox vectors, fowlpox vectors, capripox vectors, suipoxvectors, adenoviral vectors, herpes virus vectors, alpha virus vectors,baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors andpoliovirus vectors. Specific exemplary vectors are poxvirus vectors suchas vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus(MVA), adenovirus, baculovirus and the like. Pox viruses of use includeorthopox, suipox, avipox, and capripox virus. Orthopox include vaccinia,ectromelia, and raccoon pox. One example of an orthopox of use isvaccinia. Avipox includes fowlpox, canary pox and pigeon pox. Capripoxinclude goatpox and sheeppox. In one example, the suipox is swinepox.Examples of pox viral vectors for expression as described for example,in U.S. Pat. No. 6,165,460, which is incorporated herein by reference.Other viral vectors that can be used include other DNA viruses such asherpes virus and adenoviruses, and RNA viruses such as retroviruses andpolio.

The vector can encode a selectable marker. In one example, the poxvirusincludes, for example, a thymidine kinase gene (see U.S. Pat. No.6,998,252, which is incorporated herein by reference).

Viral vectors that OPG, a functional fragment, variant, or fusionprotein thereof include at least one expression control elementoperationally linked to the nucleic acid sequence encoding OPG, variantsthereof and fusion proteins. The expression control elements areinserted in the vector to control and regulate the expression of thenucleic acid sequence. Examples of expression control elements of use inthese vectors includes, but is not limited to, lac system, operator andpromoter regions of phage lambda, yeast promoters and promoters derivedfrom polyoma, adenovirus, retrovirus or SV40. Additional operationalelements include, but are not limited to, leader sequence, terminationcodons, polyadenylation signals and any other sequences necessary forthe appropriate transcription and subsequent translation of the nucleicacid sequence encoding OPG, a functional fragment, variant, or fusionprotein thereof in the host system. The expression vector can containadditional elements necessary for the transfer and subsequentreplication of the expression vector containing the nucleic acidsequence in the host system. Examples of such elements include, but arenot limited to, origins of replication and selectable markers. It willfurther be understood by one skilled in the art that such vectors areeasily constructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology,” John Wiley and Sons, New York,N.Y.) and are commercially available.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding OPG, variants thereof and fusionproteins, are known in the art. Such techniques involve, for example,homologous recombination between the viral DNA sequences flanking theDNA sequence in a donor plasmid and homologous sequences present in theparental virus (Mackett et al., 1982, Proc. Natl. Acad. Sci. USA79:7415-7419. The vector can be constructed for example by steps knownin the art, such as by using a unique restriction endonuclease site thatis naturally present or artificially inserted in the parental viralvector to insert the heterologous DNA.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation. Non-limitingexamples of suitable host cells include bacteria, archea, insect, fungi(for example, yeast), plant, and animal cells (for example, mammaliancells, such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, Jakoby and Pastan (eds),1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press,Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly usedmammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38,BHK, and COS cell lines, although cell lines may be used, such as cellsdesigned to provide higher expression desirable glycosylation patterns,or other features. As discussed above, techniques for the transformationof yeast cells, such as polyethylene glycol transformation, protoplasttransformation and gene guns are also known in the art (see Gietz andWoods Methods in Enzymology 350: 87-96, 2002).

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding OPG, a functionalfragment, variant, or fusion protein thereof, and a second foreign DNAmolecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide can becarried out by conventional means including preparative chromatographyand immunological separations. Once expressed, OPG, a functionalfragment, variant, or fusion protein thereof can be purified accordingto standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95%homogeneity are disclosed herein, and 98 to 99% or more homogeneity canbe used for pharmaceutical purposes. Once purified, partially or tohomogeneity as desired, if to be used therapeutically, the polypeptidesshould be substantially free of endotoxin.

In addition to recombinant methods, OPG, a functional fragment, variant,or fusion protein thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagent N,N′-dicyclohexylcarbodimide) are well known in the art.

Pharmaceutical Compositions and Therapeutic Methods

Pharmaceutical compositions that include OPG, a functional fragment,variant, or fusion protein thereof, or a nucleic acid encoding thesepolypeptides, can be formulated with an appropriate pharmaceuticallyacceptable carrier, depending upon the particular mode of administrationchosen.

In some embodiments, the pharmaceutical composition consists essentiallyof OPG, a variant thereof and/or a fusion protein (or a nucleic acidencoding these molecules) and a pharmaceutically acceptable carrier. Inthese embodiments, additional therapeutically effective agents are notincluded in the compositions.

In other embodiments, the pharmaceutical composition includes OPG, afunctional fragment, variant, or fusion protein thereof (or a nucleicacid encoding these molecules) and a pharmaceutically acceptablecarrier. Addition therapeutic agents, such as agents for the treatmentof diabetes, can be included. Thus, the pharmaceutical compositions caninclude a therapeutically effective amount of another agent. Examples ofsuch agents include, without limitation, anti-apoptotic substances suchas the Nemo-Binding Domain and compounds that induce proliferation suchas cyclin dependent kinase (CDK)-6, CDK-4 and Cyclin D1. Other activeagents can be utilized, such as antidiabetic agents for example,metformin, sulphonylureas (e.g. glibenclamide, tolbutamide,glimepiride), nateglinide, repaglinide, thiazolidinediones (e.g.rosiglitazone, pioglitazone), peroxisome proliferator-activated receptor(PPAR)-gamma-agonists (such as C1262570) and antagonists,PPAR-gamma/alpha modulators (such as KRP 297), alpha-glucosidaseinhibitors (e.g. acarbose, voglibose), Dipeptidyl peptidase (DPP)-IVinhibitors (such as LAF237, MK-431), alpha2-antagonists, agents forlowering blood sugar, cholesterol-absorption inhibitors,3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors(such as a statin), insulin and insulin analogues, GLP-1 and GLP-1analogues (e.g. exendin-4) or amylin. Additional examples includeimmunomodulatory factors such as anti-CD3 mAb, growth factors such asHGF, VEGF, PDGF, lactogens, and PTHrP.

The pharmaceutically acceptable carriers and excipients useful in thisdisclosure are conventional. See, e.g., Remington: The Science andPractice of Pharmacy, The University of the Sciences in Philadelphia,Editor, Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21^(st)Edition (2005). For instance, parenteral formulations usually compriseinjectable fluids that are pharmaceutically and physiologicallyacceptable fluid vehicles such as water, physiological saline, otherbalanced salt solutions, aqueous dextrose, glycerol or the like. Forsolid compositions (e.g., powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, pH buffering agents, or the like, for example sodiumacetate or sorbitan monolaurate. Excipients that can be included are,for instance, other proteins, such as human serum albumin or plasmapreparations.

In some embodiments, OPG, a functional fragment, variant, or fusionprotein thereof is included in a controlled release formulation, forexample, a microencapsulated formulation. Various types of biodegradableand biocompatible polymers, methods can be used, and methods ofencapsulating a variety of synthetic compounds, proteins and nucleicacids, have been well described in the art (see, for example, U.S.Patent Publication Nos. 2007/0148074; 2007/0092575; and 2006/0246139;U.S. Pat. Nos. 4,522,811; 5,753,234; and 7,081,489; PCT Publication No.WO/2006/052285; Benita, Microencapsulation: Methods and IndustrialApplications, 2^(nd) ed., CRC Press, 2006).

In other embodiments, OPG, a functional fragment, variant, or fusionprotein thereof is included in a nanodispersion system. Nanodispersionsystems and methods for producing such nanodispersions are well known toone of skill in the art. See, e.g., U.S. Pat. No. 6,780,324; U.S. Pat.Publication No. 2009/0175953. For example, a nanodispersion systemincludes a biologically active agent and a dispersing agent (such as apolymer, copolymer, or low molecular weight surfactant). Exemplarypolymers or copolymers include polyvinylpyrrolidone (PVP),poly(D,L-lactic acid) (PLA), poly(D,L-lactic-co-glycolic acid (PLGA),poly(ethylene glycol). Exemplary low molecular weight surfactantsinclude sodium dodecyl sulfate, hexadecyl pyridinium chloride,polysorbates, sorbitans, poly(oxyethylene) alkyl ethers,poly(oxyethylene) alkyl esters, and combinations thereof. In oneexample, the nanodispersion system includes PVP and ODP or a variantthereof (such as 80/20 w/w). In some examples, the nanodispersion isprepared using the solvent evaporation method, see for example, Kanazeet al., Drug Dev. Indus. Pharm. 36:292-301, 2010; Kanaze et al., J.Appl. Polymer Sci. 102:460-471, 2006. With regard to the administrationof nucleic acids, one approach to administration of nucleic acids isdirect treatment with plasmid DNA, such as with a mammalian expressionplasmid. As described above, the nucleotide sequence encoding OPG, afunctional fragment, variant, or fusion protein thereof can be placedunder the control of a promoter to increase expression of the molecule.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include polymer based systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems, such as lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono-di- and tri-glycerides; hydrogel release systems; silasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which OPG, a functional fragment, variant, or fusion proteinthereof, or polynucleotide encoding these polypeptides is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775; 4,667,014; 4,748,034; 5,239,660; and 6,218,371 and (b)diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,832,253 and 3,854,480. In addition, pump-based hardware deliverysystems can be used, some of which are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions, such as diabetes.Long-term release, as used herein, means that the implant is constructedand arranged to deliver therapeutic levels of the active ingredient forat least 30 days, and preferably 60 days. Long-term sustained releaseimplants are well known to those of ordinary skill in the art andinclude some of the release systems described above. These systems havebeen described for use with nucleic acids (see U.S. Pat. No. 6,218,371).For use in vivo, nucleic acids and peptides are preferably relativelyresistant to degradation (such as via endo- and exo-nucleases). Thus,modifications, such as the inclusion of a C-terminal amide, can be used.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical, inhalation, oral and suppositoryformulations can be employed. Topical preparations can include eyedrops, ointments, sprays, patches and the like. Inhalation preparationscan be liquid (e.g., solutions or suspensions) and include mists, spraysand the like. Oral formulations can be liquid (e.g., syrups, solutionsor suspensions), or solid (e.g., powders, pills, tablets, or capsules).Suppository preparations can also be solid, gel, or in a suspensionform. For solid compositions, conventional non-toxic solid carriers caninclude pharmaceutical grades of mannitol, lactose, cellulose, starch,or magnesium stearate. Actual methods of preparing such dosage forms areknown, or will be apparent, to those skilled in the art.

The pharmaceutical compositions that include OPG, a functional fragment,variant, or fusion protein thereof can be formulated in unit dosageform, suitable for individual administration of precise dosages. In onespecific, non-limiting example, a unit dosage contains from about 1 mgto about 1 g of include OPG, a functional fragment, variant, or fusionprotein thereof, such as about 10 mg to about 100 mg, about 50 mg toabout 500 mg, about 100 mg to about 900 mg, about 250 mg to about 750mg, or about 400 mg to about 600 mg. In other examples, atherapeutically effective amount of OPG, a functional fragment, variant,or fusion protein thereof is about 0.01 mg/kg to about 50 mg/kg, forexample, about 0.5 mg/kg to about 25 mg/kg or about 1 mg/kg to about 10mg/kg. In other examples, a therapeutically effective amount of OPG, afunctional fragment, variant, or fusion protein thereof is about 1 mg/kgto about 5 mg/kg, for example about 2 mg/kg. In a particular example, atherapeutically effective amount of OPG, a functional fragment, variant,or fusion protein thereof includes about 1 mg/kg to about 10 mg/kg, suchas about 2 mg/kg.

The amount of active compound(s) administered will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated. Atherapeutically effective amount of OPG, a functional fragment, variant,or fusion protein thereof can be the amount of OPG, a functionalfragment, variant, or fusion protein thereof, or a nucleic acid encodingthese molecules that is necessary to treat diabetes or affect glucosetolerance.

A therapeutically effective amount can be administered in a single dose,twice daily, weekly, or in several doses, for example daily, or during acourse of treatment. However, the therapeutically effective amount willbe dependent on the subject being treated, the severity and type of theaffliction, and the manner of administration of the therapeutic(s).Islets of β cells can also be treated prior to transplantation into asubject.

When a viral vector is utilized for administration of an nucleic acidencoding OPG, a functional fragment, variant, or fusion protein thereof,it is desirable to provide the recipient with a dosage of eachrecombinant virus in the composition in the range of from about 10⁵ toabout 10¹⁰ plaque forming units/mg mammal, although a lower or higherdose can be administered. The composition of recombinant viral vectorscan be introduced into a mammal either prior to any evidence of acancer, or to mediate regression of the disease in a mammal afflictedwith the cancer. Examples of methods for administering the compositioninto mammals include, but are not limited to, exposure of cells to therecombinant virus ex vivo, or injection of the composition into theaffected tissue or intravenous, subcutaneous, intradermal orintramuscular administration of the virus. Alternatively the recombinantviral vector or combination of recombinant viral vectors may beadministered locally by direct injection into the cancerous lesion in apharmaceutically acceptable carrier. Generally, the quantity ofrecombinant viral vector, carrying the nucleic acid sequence of thepolypeptides to be administered is based on the titer of virusparticles. An exemplary range to be administered is 10⁵ to 10¹⁰ virusparticles per mammal, such as a human.

The compositions of this disclosure that include OPG, a functionalfragment, variant, or fusion protein thereof (or nucleic acids encodingthese molecules) can be administered to humans or other animals by anymeans, including orally, intravenously, intramuscularly,intraperitoneally, intranasally, intradermally, intrathecally,subcutaneously, via inhalation or via suppository. In one non-limitingexample, the composition is administered orally. In further examples,site-specific administration of the composition can be used, for exampleby administering OPG, a functional fragment, variant, or fusion proteinthereof (or a nucleic acid encoding these molecules) to pancreas tissue(for example by using a pump, or by implantation of a slow release format the site of the pancreas). As noted above, treatment can involvedaily or multi-daily or less than daily (such as weekly or monthly etc.)doses over a period of a few days to months, or even years. In aparticular non-limiting example, treatment involves once daily dose ortwice daily dose. Islets and/or β cells can be treated prior totransplantation into a subject. The particular mode of administrationand the dosage regimen will be selected by the attending clinician,taking into account the particulars of the case (e.g. the subject, thedisease, the disease state involved, the particular treatment, andwhether the treatment is prophylactic).

The present disclosure also includes combinations of OPG, a functionalfragment, variant, or fusion protein thereof, or a nucleic acid encodingone or more of these molecules, with one or more other agents useful inthe treatment of diabetes or insulin resistance.

Anti-diabetic agents are generally categorized into six classes:biguanides; thiazolidinediones; sulfonylureas; inhibitors ofcarbohydrate absorption; fatty acid oxidase inhibitors andanti-lipolytic drugs; and weight-loss agents. Any of these agents canalso be used in the methods disclosed herein. The anti-diabetic agentsinclude those agents disclosed in Diabetes Care, 22(4):623-634, hereinincorporated by reference. One class of anti-diabetic agents of use isthe sulfonylureas, which are believed to increase secretion of insulin,decrease hepatic glucogenesis, and increase insulin receptorsensitivity. Another class of anti-diabetic agents of use the biguanideantihyperglycemics, which decrease hepatic glucose production andintestinal absorption, and increase peripheral glucose uptake andutilization, without inducing hyperinsulinemia.

In some examples, OPG, a functional fragment, variant, or fusion proteinthereof can be administered in combination with effective doses ofanti-diabetic agents (such as biguanides, thiazolidinediones, orincretins) and/or lipid lowering compounds (such as statins orfibrates). The term “administration in combination” or“co-administration” refers to both concurrent and sequentialadministration of the active agents. Administration of OPG, a functionalfragment, variant, or fusion protein thereof or a nucleic acid encodingone or more of these molecules, may also be in combination withlifestyle modifications, such as increased physical activity, low fatdiet, low sugar diet, and smoking cessation. Additional agents of useinclude, without limitation, anti-apoptotic substances such as theNemo-Binding Domain and compounds that induce proliferation such ascyclin dependent kinase (CDK)-6, CDK-4 and Cyclin D1. Other activeagents can be utilized, such as antidiabetic agents for example,metformin, sulphonylureas (e.g. glibenclamide, tolbutamide,glimepiride), nateglinide, repaglinide, thiazolidinediones (e.g.rosiglitazone, pioglitazone), peroxisome proliferator-activated receptor(PPAR)-gamma-agonists (such as C1262570) and antagonists,PPAR-gamma/alpha modulators (such as KRP 297), alpha-glucosidaseinhibitors (e.g. acarbose, voglibose), Dipeptidyl peptidase (DPP)-IVinhibitors (such as LAF237, MK-431), alpha2-antagonists, agents forlowering blood sugar, cholesterol-absorption inhibitors,3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors(such as a statin), insulin and insulin analogues, GLP-1 and GLP-1analogues (e.g. exendin-4) or amylin. In some embodiments the agent isan immunomodulatory factor such as anti-CD3 mAb, growth factors such asHGF, vascular endothelial growth factor (VEGF), platelet derived growthfactor (PDGF), lactogens, or parathyroid hormone related protein (PTHrP)

It is disclosed herein that of OPG, a functional fragment, variant, orfusion protein thereof increase beta cell survival and proliferation.The beta cell can be a human beta cell. The beta cell can be either invivo or in vitro. The human beta cells can derived from other cell typessuch as stem cells (embryonic stem cells, adult stem cells, inducedpluripotent stem cells), progenitor cells, or can be produced bytransdifferentiation from other cell types

Methods of determining or otherwise measuring beta cell proliferationare known in the art. The method can include measuring the number ofbeta cells in a sample from the subject. The method can also includemeasuring incorporation of a substance incorporated into DNA, such as,but not limited to, bromodeoxyuridine or Ki-67.

In one embodiment, a membrane permeable cell proliferation dye isutilized. In some examples, the dye is fluorescent. These dyes include,but are not limited to carboxyfluorescein diacetate, or succinimidylester. In some examples, a cell proliferation dye is used that isfluorescent and can be identified using fluorescence activated cellsorting. In some embodiments, cell viability is also assayed. There area variety of suitable cell viability assays which can be used,including, but not limited to, light scattering, viability dye staining,and exclusion dye staining.

In some embodiments, methods are provided for treating diabetes orpre-diabetes in a subject by administering a therapeutically effectiveamount of a composition including OPG, a functional fragment, variant,or fusion protein thereof, or a nucleic acid encoding one or more ofthese molecules to the subject. The subject can have diabetes type I ordiabetes type II. The subject can be any mammalian subject, includinghuman subjects. The subject can be a child or an adult. The subject canalso be administered insulin. The method can include measuring beta cellproliferation.

In some examples, the method includes selecting a subject with diabetes,such as type I or type II diabetes, or a subject at risk for diabetes,such as a subject with pre-diabetes. These subjects can be selected fortreatment.

In some examples, a subject with diabetes may be clinically diagnosed bya fasting plasma glucose (FPG) concentration of greater than or equal to7.0 millimole per liter (mmol/L) (126 milligram per deciliter (mg/dL)),or a plasma glucose concentration of greater than or equal to 11.1mmol/L (200 mg/dL) at about two hours after an oral glucose tolerancetest (OGTT) with a 75 gram (g) load, or in a patient with classicsymptoms of hyperglycemia or hyperglycemic crisis, a random plasmaglucose concentration of greater than or equal to 11.1 mmol/L (200mg/dL), or HbA1c levels of greater than or equal to 6.5%. In otherexamples, a subject with pre-diabetes may be diagnosed by impairedglucose tolerance (IGT). An OGTT two-hour plasma glucose of greater thanor equal to 140 mg/dL and less than 200 mg/dL (7.8-11.0 mM), or afasting plasma glucose (FPG) concentration of greater than or equal to100 mg/dL and less than 125 mg/dL (5.6-6.9 mmol/L), or HbA1c levels ofgreater than or equal to 5.7% and less than 6.4% (5.7-6.4%) isconsidered to be IGT, and indicates that a subject has pre-diabetes.Additional information can be found in Standards of Medical Care inDiabetes—2010 (American Diabetes Association, Diabetes Care 33:S11-61,2010, incorporated herein by reference).

In some examples, treating diabetes includes one or more of increasingglucose tolerance, decreasing insulin resistance (for example,decreasing plasma glucose levels, decreasing plasma insulin levels, or acombination thereof), decreasing serum triglycerides, decreasing freefatty acid levels, and decreasing HbA1c levels in the subject. In someembodiments, the disclosed methods include measuring glucose tolerance,insulin resistance, plasma glucose levels, plasma insulin levels, serumtriglycerides, free fatty acids, and/or HbA1c levels in a subject.

In some examples, administration of OPG a variant thereof and/or afusion protein treats diabetes or pre-diabetes by increasing glucosetolerance, for example, by decreasing blood glucose levels (such astwo-hour plasma glucose in an OGTT or FPG) in a subject. In someexamples, the method includes decreasing blood glucose by at least 5%(such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as comparedwith a control. In particular examples, a decrease in blood glucoselevel is determined relative to the starting blood glucose level of thesubject (for example, prior to treatment with OPG, a functionalfragment, variant, or fusion protein thereof). In other examples,decreasing blood glucose levels of a subject includes reduction of bloodglucose from a starting point (for example greater than about 126 mg/dLFPG or greater than about 200 mg/dL OGTT two-hour plasma glucose) to atarget level (for example, FPG of less than 126 mg/dL or OGTT two-hourplasma glucose of less than 200 mg/dL). In some examples, a target FPGmay be less than 100 mg/dL. In other examples, a target OGTT two-hourplasma glucose may be less than 140 mg/dL. Methods to measure bloodglucose levels in a subject (for example, in a blood sample from asubject) are routine.

In some embodiments, the disclosed methods include treating a subjectwith diabetes by increasing beta cell proliferation. In some examples,administration of OPG, a functional fragment, variant, or fusion proteinthereof treats diabetes by increasing beta cell number in a subject, forexample increasing beta cells proliferation by at least 5% (such as atleast 10%, 15%, 20%, 25%, 30%, 35%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300% or more) as compared with a control (such as the subject prior toadministration of the OPG, variant thereof or fusion thereof). Methodsof determining measuring beta cell proliferation are known in the art.The method can include measuring the number of beta cells in a sample,such as a biopsy, from the subject. The method can also includemeasuring incorporation of a substance incorporated into DNA, such asbromodeoxyuridine or Ki-67.

In other embodiments, the disclosed methods include comparing one ormore indicator of diabetes (such as glucose tolerance, triglyceridelevels, free fatty acid levels, or HbA1c levels) to a control, whereinan increase or decrease in the particular indicator relative to thecontrol (as discussed above) indicates effective treatment of diabetes.The control can be any suitable control against which to compare theindicator of diabetes in a subject. In some embodiments, the control isa sample obtained from a healthy subject (such as a subject withoutdiabetes). In some embodiments, the control is a historical control orstandard reference value or range of values (such as a previously testedcontrol sample, such as a group of subjects with diabetes, or group ofsamples from subjects that do not have diabetes). In further examples,the control is a reference value, such as a standard value obtained froma population of normal individuals that is used by those of skill in theart. Similar to a control population, the value of the sample from thesubject can be compared to the mean reference value or to a range ofreference values (such as the high and low values in the reference groupor the 95% confidence interval). In other examples, the control is thesubject (or group of subjects) treated with placebo compared to the samesubject (or group of subjects) treated with the therapeutic compound ina cross-over study. In further examples, the control is the subject (orgroup of subjects) prior to treatment.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 OPG is a Novel Downstream Target of Lactogens in theβ Cell

Lactogens play a crucial role in β-cell physiology, with known salutaryeffects on β-cell growth, function, and survival. Mouse placentallactogen 1 (mPL1) expressed under the rat insulin promoter (RIP)enhances β-cell proliferation, survival and mass in the RIP-mPL1transgenic (TG) mice. PCR-array analysis in islets from RIP-mPL1 TG andnormal (NL) mice have identified OPG as a novel downstream target oflactogens. OPG was the most consistently highly expressed gene in theislets of TG versus NL mice in this analysis (FIG. 2A). These resultswere confirmed by real-time PCR analysis in TG versus NL islets (FIG.2B) as well as in INS-1 cells acutely treated with prolactin (Prl, FIG.2C). Lactogens also increase OPG protein levels in mouse islets (FIG.2D) and INS-1 cells (FIG. 2E) as seen by Western blot analysis. This isthe fourth model of rodent beta cell expansion in which OPG expressionis up-regulated, suggesting a correlation between OPG expression andincreased beta cell proliferation.

Example 2 Expression of RANK and Death Receptors in Rodent Islets andINS-1 Cells

OPG does not have any intrinsic signaling capabilities; it achieves itsfunctions in other cell types through the modulation of two specificligand/receptor systems, RANKL/RANK and TRAIL/DRs (FIG. 3A). Bothligands, RANKL and TRAIL, are expressed in pancreatic islets. It wasexamined whether the receptors for these molecules are expressed inrodent islets and in INS-1 cells by real-time PCR using species-specificprimers. Results showed that the mRNA of RANK receptor (FIG. 3B) as wellas the DRS receptor (FIG. 3C), (that bind RANKL and TRAIL ligands,respectively) are expressed in mouse islets as well as in INS-1 cells,suggesting that OPG has the potential to act through either pathway inthe β-cell.

Example 3 OPG Enhances Rodent β-Cell Proliferation In Vivo

It was hypothesized that OPG will enhance β-cell replication. To testthis hypothesis, subcutaneous (s.c.) daily injections of recombinantmouse-Fc chimera OPG (mOPG, R&D Systems) were administered at severaldifferent doses (2, 10, 50, 250, 500, 1000, and 2000 ng/g body weight,or saline as control, for 7 days in 8-week old C57BL6 male mice, andmeasured β-cell proliferation by insulin (green)-bromodeoxyuridine(BrdU) (red) co-staining (FIG. 4A). There was no change in body weightand blood glucose in any of the groups. There was trend to an increasein plasma insulin at higher doses, but it was not statisticallysignificant. An intra peritoneal glucose tolerance test (IPGTT) wasperformed. All groups were similar to vehicle-treated, although therewas a slight improvement in glucose clearance with the higher doses(50-2000 ng). There was no change in beta cell apoptosis in the groups.However, with regard to beta cell proliferation, a 2-3 foldnon-significant increase was demonstrated at the lower doses (2-250 ng),and a significant 4-7 fold increase was demonstrated at the higher(500-2000 ng) doses.

A mOPG dose response (0, 10, 50, and 500 ng) study for 30 days in vivowas also performed in 8-week old C57Bl6 mice (n=4 mice/group). Bodyweight and blood glucose were measured. An IPGTT was performed at 1 and4 weeks, and an insulin tolerance test (ITT) was performed at 3 weeks.There was no change in these values. Beta cell proliferation wasmeasured at 4 weeks. An increase in beta cell proliferation at 50 and500 ng doses, which was significant at the 500 ng dose.

Generally the data showed that at the lower doses (2-250 ng/g) OPGincreases beta cell proliferation, although not significantly, by2-3-fold over control. At the higher doses (500-2000 ng/g) OPGsignificantly and markedly induces rodent β-cell proliferation by4-8-fold over control at day 7 in vivo (FIG. 4B) (n=3-8 mice/group). OPGtreatment did not negatively affect blood glucose levels nor did itsignificantly change body weight of these mice. Longer (30 day)treatment with mOPG (10-500 ng/g body weight) also resulted in asignificant increase in β-cell proliferation at the higher 500 ng/g dose(FIG. 4C) (n=4-5 mice/group).

Example 4 OPG Induces Human β-Cell Replication In Vitro

To determine if the in vivo proliferative action of OPG (FIG. 4) is aresult of a direct effect on the β-cell, and whether this can bereproduced in human β-cells in vitro, additional studies were performed.Human islet cell cultures were treated with different doses, 25, 50,100, and 200 ng/ml of recombinant human-Fc chimera OPG (hOPG), orvehicle (veh), for 24 h and quantified β-cell proliferation byBrdU-insulin co-staining (FIG. 5A). There was a significant increase inhuman β-cell replication with the three higher doses of hOPG (FIG. 5B).Beta cell replication was also measured using a different assay,insulin-Ki67 co-staining, of human islet cell cultures treated with 100ng/ml of hOPG or vehicle as control for two different time-points, 24 hand 72 h. OPG significantly enhanced human beta cell proliferation atboth time points with this method (FIG. 5C). This result alsodemonstrates that OPG has a proliferative effect in aged β-cells, as theaverage age of the human islet donors was 50.3±3.3 years.

Example 5 OPG Improves Human α-Cell Survival

OPG could protect human β-cells against GLT-induced cell death. Toexamine this, human islet cell cultures were treated with 200 ng/ml ofhOPG for 36 h±GLT (25 mM glucose and 0.5 m M palmitate), and β-celldeath quantified by TUNEL insulin co-staining (FIG. 6A). Human β-cellswere significantly protected against GLT-induced cell death inhOPG-versus vehicle-treated cells (FIG. 6B). It was determined if OPGenhanced survival of human β-cells against cytokine induced cell death.The results indicate that different doses (25-200 ng/ml) of hOPG protecthuman β-cells against cytokine-induced cell death (FIG. 6C).

The studies disclosed herein identify OPG as a novel, biologicaldownstream target of lactogens in β-cells. Proliferative effects of OPGwere observed in human β-cells in vitro. This effect clearly suggeststhat OPG has regenerative potential to enhance endogenous functionalβ-cell mass under stress-induced conditions. The pro-survival effects ofOPG against cytokines in human islets (FIG. 6C), strongly suggest thatOPG treatment will protect/delay the onset of Type 1 diabetes. OPG canbe administered repeatedly, such as for months (for example 1-12months), or years (for example at least 1, 2, 3, 4 or 5 years).

Example 6 Systemic OPG Administration on Rodent Beta Cell Growth,Function, and Survival Under Basal, Aging, Regenerative, and Type 1Diabetes Conditions

The effect of different doses of mouse OPG administration on glucose and(3-cell homeostasis in young adult mice is determined. Aging is known toimpair basal β-cell proliferation in rodents and in humans. The normalinduction in proliferation caused by injury is also impaired in β-cellsof older mice. Despite this there have been very few studies thatdirectly address the regenerative therapeutic potential of growthfactors on the “aged” β-cell.

OPG can have the capacity to enhance basal β-cell proliferation in oldermice. The ability to stimulate β-cell proliferation in human donors withan average age of 50.3±3.3 years, by three different doses of OPG (FIG.5), suggests that OPG will induce β-cell proliferation in older mice.Regeneration of β-cell mass in patients with diabetes will requires anagent that, when administered acutely, stimulates β-cell hyperplasia inthe already injured endocrine pancreas without negatively affectingglucose homeostasis.

The regenerative potential of OPG using double transgenic mice in whichthe diphtheria toxin (DT) receptor (DTR) is expressed specifically inthe β-cell, and cell death is induced upon acute injection of the DTligand. This model for regeneration, is inducible and reversible.

Different doses of systemic OPG administration on glucose and β-cellhomeostasis are tested in young adult mice. Eight week old C57BL6 malemice are injected daily s.c. with mouse recombinant OPG (mOPG, from R &D Systems) at the following doses: 10, 50, 100, 200, 500, and 1000 ng/gbody weight, or an equal volume of saline as control. The effects ofthese doses at two different time points, 7 and 30 days, are examinedsee FIG. 7.

9-month-old C57BL6 male mice from JAX labs are first aged to 12 months,and then subjected to a similar experimental design, mOPG doses (10-1000ng/g), and outcome measurements as outlined and FIG. 7.

The regenerative potential of OPG administration is evaluated in thedouble transgenic DTR mouse model. This system has been widely usedpreviously (Buch T, et al., Nat Methods 2:419-426, 2005; Criscimanna A,et al. Gastroenterology 141:1451-1462, 2011.) and the two transgeniclines required, R26^(DTR) and the RIP-Cre mice are available in JAXlabs. Briefly, RIP-Cre mice are bred with the R26^(DTR) mice to obtaindouble transgenic hemizygous RIP-Cre/R26^(DTR) mice that expresses DTRin their β-cells due to removal of a floxed stop signal in front of theDTR cDNA. Eight week old male RIP-Cre/R26^(DTR) or control R26^(DTR)mice will be injected intraperitoneally (ip) daily for 5 days withdiphtheria toxin (DT) 0.5 ng/g body weight to induce destruction of theDTR-expressing β-cell. After the last DT injection, the mice are treatedwith vehicle or mOPG and body weight, blood glucose, plasma insulin,IPGTT, and ITT measured on the days indicated in FIG. 8 A,B. Mice aresacrificed at one and four weeks when β-cell parameters including,proliferation, death, and mass will be measured.

To determine the effect of OPG administration on glucose and β-cellhomeostasis in NOD/Ltj mice, a model of Type 1 diabetes is utilized.NOD/Ltj female mice, a well characterized model of autoimmune Type 1diabetes, obtained from JAX labs is used for these studies. 14 week oldpre-diabetic NOD/Ltj female mice are injected with saline (as control)or mOPG for 1, 2, and 6 weeks, and glucose and β-cell homeostasis isassessed. It is determined whether OPG treatment delays or prevents theoccurrence of diabetes in these mice over the 6-week period. At the 1and 2 week earlier time points, the rate of β-cell death and insulitisis examined.

Example 7 The Direct Effect of OPG on Human and Rodent Beta CellProliferation, Survival, and Function In Vitro

To Examine the Effect of OPG on Rodent and Human β-Cell Proliferation InVitro

The data presented herein shows that different doses of human OPG (hOPG)can induce β-cell proliferation in human islet cell cultures from donorswith an average age of 50.3±3.3 years (FIG. 5). The proliferationresponse at different doses of 0.05-5.0 μg/ml of hOPG and theproliferative response at different times (1-7 days) after treatment isexamined, fresh hOPG peptide is added daily to the human islet cellculture. Human β-cell proliferation is measured by insulin-BrdUco-staining, shown in FIG. 5.

To Examine the Effect of OPG on Rodent and Human β-Cell Survival InVitro

Glucolipotoxicity (GLT), cytokines, and endoplasmic reticular(ER)-stress are causes of β-cell death in diabetes. For additionalconfirmatory sludies, the pro-survival effects of hOPG against thesecell death inducers on human β-cells in vitro, is characterization interms of duration and dose-dependency. The effect of different doses ofhOPG, ranging from 0.05-5.0 μg/ml, is tested. Similar to theexperimental design in FIG. 6, human islet cell cultures are pre-treatedin the absence of serum with hOPG and subsequently cell death is inducedeither by GLT (25 mM glucose and 0.5 mM palmitate), human cytokines(IL-1β, TNF-α, and IFN-γ), or thapsigargin (0.1-1.0 μM) to induceER-stress, for 24-96 h, and β-cell death quantified by TUNEL-insulinco-staining. Similar analysis is performed on mouse islets using mouseOPG. GLT and cytokine studies in human islets are done (FIG. 6).

The Effect of OPG on Rodent and Human β-Cell Function In Vitro.

The effect of hOPG on insulin expression and glucose-stimulated insulinsecretion (GSIS) response in human islets was assessed. Whole humanislets treated with vehicle or different concentrations of hOPG(0.05-5.0 μg/ml) for different times (24-96 hrs) were analyzed forinsulin expression at the mRNA level by real time PCR, and at theprotein level by an RIA for human insulin, and the secretory responseassessed by GSIS. OPG did not change GSIS response in human islets.

In certain embodiments, a subject has been determined to have or to beat risk or developing diabetes. For example someone determined to haverisk factors (such as obesity) or laboratory evidence (such as anelevated serum glucose or hemoglobin A1c level) of prediabetes ordiabetes. The OPG is then administered to the subject for months oryears. Laboratory evidence or the condition can be assessed, for exampleby treating the subject's fasting glucose or HbA1c levels.

Example 8 The Effect of OPG on Human Beta Cells In Vivo

The studies above clearly show that OPG enhances survival andproliferation of human β-cells in vitro (FIGS. 5 and 6). The diabeticnon-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, forboth mouse and human islets is used to examine the effect of hOPG onhuman islet transplant outcomes in vivo.

The Effect of hOPG in the Setting of a Suboptimal Marginal Mass Model ofHuman Islet Transplants in Diabetic NOD/SCID Mice.

NOD/SCID male mice are made diabetic by injection of STZ (2 doses of 125mg/Kg-body weight given 20 hr apart). Mice with random non-fasted bloodglucose>300 mg/dl for three consecutive days are considered diabetic. Aninsufficient number (1500) of human islet equivalents (IEs, 1 IE=125 μmdiameter islet), less than the minimal number of islets required tocause euglycemia, are handpicked and transplanted under the kidneycapsule of diabetic immunodeficient NOD/SCID mice, and vehicle or hOPGis administered in the mice from the day of the transplant up to 8 weeks(n=8-10/group). Glucose and β-cell homeostasis, including blood glucose,plasma insulin (mouse and human, using specific RIA kits, IPGTT, β-cellproliferation and death in the islet grafts and in the pancreas aremeasured at early (1 week) and late (8 weeks) time-points aftertransplantation. To ensure that euglycemia observed after thetransplantation is a result of a functioning human islet graft,transplanted mice undergo unilateral nephrectomy at the end of 8 weeks,and blood glucose is measured daily for the next 7 days. The kidneyswith the grafts are stained for insulin to determine the extent ofβ-cell area present at the graft site 8 weeks after the transplant.

Example 9

Human islet cell cultures (n=3-5) were treated with 25-200 ng of OPG inthe presence of bromodeoxyuridine (BrdU) and human beta cellproliferation was assessed by insulin-BrdU co-staining. There was asignificant 2.5-3.0 fold increase in human beta cell proliferation withthe higher OPG doses. In addition, human islet cell cultures (n=3-5)were treated with 25-200 ng/ml of human recombinant OPG (hOPG) andβ-cell proliferation was assessed using Ki-67 co-staining with insulin.There was a significant (3-4-fold) increase in human β-cellproliferation with the higher hOPG doses. This confirmed the observationof the pro-proliferative effect of hOPG on human β-cell proliferation byBrdU staining.

It was examined if OPG had a direct effect on human beta cell survivalagainst glucolipotoxicity (GLT) and cytokines. OPG significantlyameliorated by ˜45% and ˜50%, the 3.5-fold increase in GLT andcytokine-induced human beta cell death, respectively, as measured byinsulin and TUNEL co-staining (n=3), demonstrating a direct pro-survivaleffect of OPG on human beta cells. These studies reveal OPG to be anovel downstream target of lactogens, which can independently anddirectly enhance human beta cell proliferation and survival.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

The invention claimed is:
 1. A method for treating a subject with Type 1diabetes or prediabetes, comprising: selecting a subject with Type 1diabetes or prediabetes; and administering to the subject atherapeutically effective amount of (a) a wild type humanosteoprotegerin, an osetoprotegerin polypeptide comprising the aminoacid sequence set forth as SEQ ID NO: 1, or an osetoprotegerinpolypeptide comprising the amino acids 22-401 of the amino acid sequenceset forth as SEQ ID NO: 1, or (b) a fusion protein thereof that bindsreceptor activator of nuclear factor kappa-B ligand (RANKL), therebytreating Type 1 diabetes or prediabetes in the subject or inhibitingrecent onset of Type 1 diabetes in the subject.
 2. The method of claim1, wherein the subject is human.
 3. The method of claim 1, wherein thesubject has Type 1 diabetes, and wherein the method treats the Type 1diabetes in the subject.
 4. The method of claim 1, comprisingadministering to the subject a therapeutically effective amount of theosteoprotegerin.
 5. The method of claim 4, wherein the osteoprotegerincomprises the amino acid sequence set forth as SEQ ID NO:
 1. 6. Themethod of claim 1, comprising administering to the subject atherapeutically effective amount of the fusion protein, wherein thefusion protein comprises an immunoglobulin Fc.
 7. The method of claim 1,further comprising measuring beta cell function of the subject.
 8. Themethod of claim 7, wherein the method comprises measuring glucosetolerance, measuring plasma glucose level, measuring plasma insulinlevel, measuring serum triglycerides, measuring a free fatty acid level,and/or measuring hemoglobin (Hb)A1c level.
 9. The method of claim 1,further comprising measuring glucose tolerance, insulin resistance,plasma glucose levels, plasma insulin levels, serum triglycerides, freefatty acids, and/or HbA1c levels in a sample from the subject.
 10. Themethod of claim 1, further comprising administering an additionaltherapy for modulating body weight or treating diabetes to the subject.11. The method of claim 10, wherein the additional therapy comprises alifestyle modification.
 12. The method of claim 10, wherein theadditional therapy is insulin.
 13. The method of claim 1, wherein thesubject is a child.
 14. The method of claim 1, wherein the subject is anadult.
 15. The method of claim 1, comprising administering to thesubject a therapeutically effective amount of the osetoprotegerinpolypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1or the osteoprotegerin polypeptide comprising amino acids 22-401 of SEQID NO:
 1. 16. The method of claim 1, comprising administering to thesubject a therapeutically effective amount of the wild-type humanosteoprotegerin.